The basic Helix-Loop-Helix-Per/Arnt/Sim (bHLH-PAS) family is a major group of transcription factors in mammals. Functional heterodimers in this family form via a tissue-restricted subunit, such as the hypoxia- inducible factors (HIFs), neuronal PAS (NPAS) proteins, or aryl hydrocarbon receptor (AHR), and a common dimerization subunit, such as aryl hydrocarbon receptor nuclear translocator (ARNT). Family members contain tandem PAS domains and a DNA-binding domain. PAS domains have the potential to act as molecular sensors and transmit signals to regulate the activities of these transcription factors. The HIF subgroup within the family regulates genetic programs critical in human tumor initiation, progression, invasion and metastasis. Several NPAS proteins have been genetically linked to human neuropsychiatric disorders. AHR recognizes and responds to a spectrum of xenobiotic molecules and drives gene programs necessary for detoxification. Our current knowledge about the architectures of these heterodimers, their ligand-binding pockets, and ligand- responsive activities is limited. Therefore, we have begun to conduct detailed structural investigations into several bHLH-PAS heterodimers in complexes that include bound DNA and small-molecule ligands. Our proposed structural characterizations will be complemented with chemical biology and cell-based studies to deepen our understanding of structure-function relationships in this family. The overall goals of this proposal include characterizing distinct bHLH-PAS heterodimers by X-ray crystallography, interrogating the location and composition of their ligand-binding pockets, identifying new tool compounds, and examining how ligands and disease-linked mutations manifest their actions through protein architectures.